Resistance to
paclitaxel-based
therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired
paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a
cancer cell line to
paclitaxel in the presence of the
P-glycoprotein reversal agent,
CL-347099. The epidermoid
tumor line KB-3-1 was exposed to increasing concentrations of
paclitaxel and 5 micromol/L
CL-347099 for up to 1 year. Cells grown in 15 nmol/L
paclitaxel plus
CL-347099 (KB-15-PTX/099) developed 18-fold resistance to
paclitaxel and were dependent upon
paclitaxel for maximal growth. They grew well and retained resistance to
paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to
docetaxel, the novel
taxane MAC-321, and
epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents
vinblastine, dolastatin-10, and
HTI-286. KB-15-PTX/099-resistant cells did not overexpress
P-glycoprotein nor did they have an alteration of [14C]
paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to
glutamate substitution in class I (
M40)
beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized
tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of
paclitaxel makes contact with Asp26 of
beta-tubulin, suggesting a
ligand-induced mutation. Optimized model complexes of
paclitaxel,
docetaxel, and
MAC-321 in
beta-tubulin show a novel hydrogen bonding pattern for the
glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the
paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of
tubulin in microtubules and reflected by less acetylated
tubulin. These results suggest that a mutation in
tubulin might affect microtubule stability as well as
drug binding and contribute to the observed resistance profile.